In International Publication number WO 2014/155381 for “Method and Apparatus for Bacterial Monitoring”, there is described an apparatus and method for detection of the concentration of bacteria and cells. A schematic illustration of a system using described in that publication is shown in FIG. 1. The system incorporates a substrate 20 with an ordered array of wells or pores having diameters to fit the size of the targets. The substrate may be a periodic macro-PSi array structure (MPSiAS) illuminated with a broadband, white light source 25, which generates a large illumination spot on the substrate, thereby illuminating a substantial portion of the substrate. The drawing of FIG. 1 shows a single ray from the source 25 and reflected back from a pore region of substrate to the single detector 26, which, in the example system of FIG. 1, is positioned in the same housing as the illuminating source unit. It is to be understood though that the optical illuminating and detection system covers all or a major part of the substrate. The host analyte 24 whose bacterial content is to be measured, is flown across the surface 21 of the substrate, in a direction essentially parallel to the surface of the array, and the bacteria in the host analyte enter the wells of the substrate by random motion away from the direction of flow. The reflected light diffracted from either the whole of the substrate, or a substantial part of it, is detected 26 and spectrally analyzed 27, 28 to provide the effective optical depth of the wells. Fast Fourier Transform analysis 28, 29 may be used for the optical analysis, and the amplitude of the reflected interference light at the detected effective optical depth provides a measure of the bacterial filling factor of the wells. Entry of target elements into wells is detected by the change in the effective optical depths of the wells. The detection may be performed in real time, such that production line bacterial monitoring may be achieved.
However, the apparatus described in that publication has a number of operational disadvantages, as a result of which, the lowest concentration of bacteria that can be measured within a reasonable time is limited. The apparatus has been shown to be capable of bacterial detection down to a concentration of approximately 106 cells/ml of live bacteria. The requirements of both the food industry and the water industry are, however, as low as 100 cells/ml., or even lower. This may make that device commercially disadvantaged.
In WO 2014/155381, since the liquid sample flows over and essentially parallel to the surface of the Macro-Porous Silicon Array Structure (MPSiAS), the bacteria can enter the pores and collect there by actively directing themselves, using their motility, into the pores. For a flow direction parallel to the surface of the array, motion of the bacteria into the pores is, in the absence of any external; influence, such as a food concentration, only achieved by the self-motion of the bacteria. Such self-motion is known to be randomly directed with an average path of the order of 30μ before the bacteria change direction, which occurs every 1 sec. or so. This means that bacteria floating in the solution further than 30μ from the surface of the array, will have a significantly lower probability of entering the pores.
The rate of bacteria entry into the pores can be calculated by:Sin=∫Q(I)*P(I)  (1)where Sin is the rate of bacteria going into the pores,Q(I) is the concentration of bacteria at a distance I from the surface of the array, andP(I) is the probability of the bacteria, at a distance I, to enter into a pore.
The integral is taken from I=0 at the array surface to I=∞.
For example, if the bacterium is under no external influence that would offer it a proffered direction in which to move, i.e. that its motion is completely random, the chance that it will move in a given direction is ¼π. The chances of a bacterium, located 30μ above a pore of dimension 2μ×2μ, to enter the pore, would therefore beP(30μ)=(2μ/30μ)2/4π  (2)
However, in the same way as bacteria enter the pores, they also have a chance to leave the pores, whether by randomly flowing back into the fluid flow outside of the pores or by actively moving themselves out.
The rate of bacteria exiting is given by:Sout=∫Q′(I)*P′(I)  (3)where Sout is the rate of pore emptying,Q′(m) is the concentration of bacteria in the pore at a depth m from the top of the pores andP′(m) is the probability of the bacteria, at a depth m, to exit a pore.
The integral is taken from m=0 at the array surface, to m=D, where D is the average pore depth.
The filling rate of the bacteria is:S=Sin−Sout  (4)
The optical signal, as described in the WO 2014/155381 reference, is the interference pattern generated between the collective reflection of broadband light from the bottom surfaces of all the pores, included within the light beam, and the surfaces of the substrate between the pores. When pores start filling with bacteria, the collective reflection pattern changes from a pattern of EOT(n) pores filled just with the sample solution (where n is the number of pores included in the light beam) to:EOT=EOT′(m)+EOT(n−m)  (5)where EOT′(m) is the reflection coming from m pores filled with bacteria. The greater the number of pores occupied with bacteria, the more the EOT changes, until almost all the pores are filled, and no further change of EOT is expected. The rate of change is proportional to the occupancy rate S and obviously decreases as the concentration Q decreases. For lower concentrations, using the apparatus of the WO 2014/155381 reference, it transpires that the EOT change rate is so low that it is not easily detected within a practical time frame. Using the WO 2014/155381 prior art system, because of the integrating nature of the detection process, it may be necessary to reach a bacterial filling factor of several percent before the change in bacterial concentration is quantitatively detected in the spectral analysis of the integrated light reflected from the whole illuminated region of the substrate. Since on-line monitoring of processes, such as on a food production line, require the detection of bacterial concentrations at levels lower than 102 cells/ml which is four orders of magnitude or more below the level of detection achieved by the WO 2014/155381 system, and in a time frame short enough not to result in undue wastage should a contamination be detected, the level of sensitivity provided by the prior art system may be insufficient for such use.
There therefore exists a need for a method and apparatus to increase detection sensitivity of the bacteria, and thus to overcome at least some of the disadvantages of prior art systems and methods.
The disclosures of each of the publications mentioned in this section and in other sections of the specification, are hereby incorporated by reference, each in its entirety.